Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressiveness

Triple-negative breast cancer (TNBC) is characterized by excessive accumulation of tumor-infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs consist of a heterogeneous population with high plasticity and are associated with tumor aggressiveness and poor prognosis. Moreover, breast cancer cells can secrete factors that influence TAM polarization. Therefore, this study aimed to evaluate the crosstalk between cancer cells and macrophages in the context of TNBC. Cytokine-polarized M2 macrophage were used as control. Distinct from the classical M2 macrophage, TAMs generated from TNBC-conditioned media upregulated both M1- and M2-associated genes, and secreted both the anti-inflammatory cytokine interleukin IL-10 and the proinflammatory cytokine IL-6 and tumor necrosis factor- α. Theses TNBC-induced TAMs exert aggressive behavior of TNBC cells. Consistently, TCGA and MTABRIC analyses of human breast cancer revealed upregulation of M1- associated genes in TNBC comparing with non-TNBC. Among these M1-associated genes, CXCL10 and IL1B were revealed to be independent prognostic factors for disease progression. In conclusion, TNBC cells induce macrophage polarization with a mixture of M1 and M2 phenotypes. These cancer-induced TAMs further enhance tumor cell growth and aggressiveness.


Introduction
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, characterized by negative expression of the estrogen receptor, progesterone receptor, and HER2. Patients with TNBC have a relatively poor prognosis, owing to an aggressive tumor behavior and the lack of molecular targets for therapy [1,2]. In addition, TNBC features a unique microenvironment distinct from other breast cancer subtypes, including a high level of tumor-infiltrating lymphocytes and tumor-associated macrophages (TAMs) [3]. TAMs originate from peripheral blood monocytes and differentiate into macrophages following recruitment to tumor sites. Accumulated evidence suggests that TAMs are a heterogeneous and plastic population, in which polarized TAMs can be identified as M1-and M2-like macrophages [4]. M1-like macrophages are known to release proinflammatory cytokines and chemokines such as tumor necrosis factor (TNF)-α, interleukin [IL]-1β, and CXCL10 and exert antitumor activity, whereas M2-like macrophages produce anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 and are associated with tissue remodeling, angiogenesis, and immune suppression, leading to tumor development [5,6]. Generally, TAMs exhibit characteristics similar to those of anti-inflammatory M2 macrophages, which enhance the aggressive features of several cancers [7]. Primary TNBC tissues express high levels of CD163+ M2-like macrophages, and the expression of CD163+ in the tumor stroma is associated with the absence of hormone receptors and increased aggressive features of breast cancer [8,9]. Previous studies have also reported that TNBC cells educate primary monocytes toward the M2-like macrophage phenotype by secreting high amounts of monocyte colony-stimulating factor (M-CSF) [8,10]. By contrast, TAMs can secrete soluble factors including growth factors, cytokines, and chemokines such as transforming growth factor [TGF]-β, vascular endothelial growth factor, M-CSF, IL-10, and CXCL10 to induce tumor progression and metastasis [11][12][13][14].
In this study, we aimed to evaluate the crosstalk between TNBC cells and macrophages. We found that soluble factors obtained from a TNBC cell line modulate monocytes toward a mixed population of M1-and M2-like macrophages. Unlike classical M2-polarized macrophage, these TAMs show an upregulation of M1-associated genes and secrete the proinflammatory cytokine IL-6. TNBC cells co-cultured with TAMs exhibited increased cell proliferation and migration. Thus, our results support the complex interactions between macrophages and TNBC in tumor development.

Cell culture
The protocol and written informed consent for obtaining peripheral blood from healthy donors were approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (IRB NO.437/62). The MDA-MB-231, MDA-MB-468, and MCF-7 human breast cancer cell lines and THP-1 monocytic cell lines, obtained from the American Type Culture Collection (ATCC1, VA, USA), were grown in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, NY, USA) and Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco), respectively, at 37˚C in a humidified incubator with 5% carbon dioxide. Both media were supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1X GlutaMAX (Gibco).

Macrophage differentiation
THP-1 cells were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, Saint Louis, MO, USA) in RPMI medium for 24 h, followed by a 24-h rest period in fresh RPMI-1640 media to allow differentiation into macrophages. For the generation of TAMs, THP-1-derived macrophages were treated with 2 mL of conditioned media (CM) from breast cancer cells for 48 h. For M2-like macrophage polarization, IL-4 and IL-13 were added to fresh RPMI media for 48 h at a final concentration of 20 ng/mL and 20 ng/mL, respectively.

Breast cancer cell CM preparation
The TNBC cell lines MDA-MB-231 and MDA-MB-468 or the hormone-positive breast cancer cell line MCF-7 were seeded at 5 × 10 5 cells per well in a 6-well tissue culture plate for 24 h.
The cultured supernatant was removed and replaced with fresh RPMI media. To remove dead cells, the cell-free supernatant was collected after 24 h of incubation and centrifuged at 400 × g for 5 min. Fresh CM was prepared for each experiment.

Quantitative reverse-transcription polymerase chain reaction assay
Total RNA was extracted with the RNeasy Mini kit (QIAGEN, Hilden, Germany). RNA (1μg/ sample) was reverse transcribed to yield cDNA using the QuantiTect reverse-transcription kit (QIAGEN). The synthesized cDNA was used as the template to measure the relative expressions of these genes: CXCL10, IL-1B, TNF, TGFB1, TGFBR2, CCL22, CCL18, and IL10. Quantitative reverse-transcription polymerase chain reaction was performed with StepOnePlusTM (Applied Biosystems, MA, USA) using SYBR green (Applied Biosystems). The mRNA expressions of the candidate genes were normalized to that of the reference gene human β-actin and calculated as 2 −ΔΔCt . The following primers were used in the amplification:

Transwell co-culture assay
THP-1 cells were seeded and stimulated with PMA at a cell density of 5 × 10 5 cells/well on Transwell inserts (0.4 μm pore size, Costar 3450; Corning, Inc., NY, USA), followed by a 24-h rest period in fresh RPMI-1640 media. The THP-1 stimulated cells were then incubated with

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either IL-4/IL-13 or conditioned media harvested from MDA-MB-231 cells for 48 h to generate cytokine-polarized M2 and TAMs as mentioned earlier. Subsequently, the Transwell inserts containing the THP-1-derived macrophage were placed with MDA-MB-231 cells that were pre-seeded overnight at a cell density of 5 × 10 5 cells/well in a Transwell-suitable 6-well plate. After co-culturing for 72 h, the Transwell inserts were removed, and the MDA-MB-231 cells were harvested and subjected to cell proliferation and migration assays.

Cell proliferation assay
MDA-MB-231 cells were harvested after the Transwell co-culture assay for the measurement of cell proliferation. MDA-MB-231-treated cells were seeded in a 96-well plate at a concentration of 5 × 10 3 cells/well in 100 μl of DMEM. The cell proliferation reagent WST-1 (Sigma-Aldrich) was added at 10 μl/well, and the plate was incubated for 4 h. Cell viability was measured at 24, 48, and 72 h, according to the manufacturer's instructions.

Cell migration assay
MDA-MB-231 cells were harvested after the Transwell co-culture assay and seeded into 96-well plates at a density of 5 × 10 4 cells/well. After overnight incubation, a wound was created by scratching the cell monolayer using a 10-μL pipette tip. The remaining cells were washed thoroughly with PBS to remove cellular debris and incubated at 37×C in serum-free media. The wound space was photographed under a phase-contrast microscope (IX51 with DP70, Olympus, Tokyo, Japan) at 0, 6, 12, and 24 h. The total wound area at each time point was determined using ImageJ software and presented as the relative value to 0 h, as previously described [15].

Cytokine measurement
THP-1-derived macrophages were treated with CM from breast cancer cells or cytokines IL-4/ IL-13 for 48 h, as described earlier. The cultured supernatant was removed, washed with PBS, and replaced with fresh RPMI media. After 24 h, to remove dead cells, the cultured supernatant was collected and centrifuged at 400 × g for 5 min. The cultured supernatant collected from THP-1 cells was stimulated with PMA for 24 h, followed by fresh RPMI media for 24 h that served as control. For quantitative detection of cytokine proteins, cell-free supernatant was assayed using the BD™ Cytometric Bead Assay (Becton Dickinson, NJ, USA), including IL-4, IL-10, TNF-α, and IL-6. The assay was performed according to the kit's instruction manual provided by the manufacturer.

TCGA and METRABRIC Datasets analysis
Gene expression analyses in primary breast cancer tissues and patient clinical data from METABRIC [16] and TCGA [17] were retrieved from cBioPortal [18]. Boxplot analysis was used to compare the mRNA level between two cancer types, TNBC and non-TNBC, categorized by ER, PR, and HER2 status. The number of tissues analyzed in all boxplots was 1,904 in METABRIC (1605 non-TNBC, 299 TNBC) and 1,093 in TCGA (978 non-TNBC, 115 TNBC). Kaplan-Meier survival analysis was performed using METABRIC study. Nine hundred fiftytwo patients with available data of gene expression and time to disease-free were included. The mRNA expression was categorized as low and high levels comparing to median expression. Logrank test p-value was used for survival analysis. Cox regression model was used for univariate and multivariate analyses of gene expression level, pathological stage (0-IV), and cancer type (TNBC, non-TNBC). Boxplot and survival analyses were generated using R program.

Statistical analysis
Results were generated from three or more independent experiments. Values are presented as mean ± standard error of the mean (SEM). Data with three or more groups were analyzed statistically by one-way analysis of variance and Tukey's multiple comparisons test. For comparison between two groups, a two-tailed unpaired t-test was used. A value of p � 0.05 was accepted as significant ( � p < 0.05; �� , p < 0.01; ��� , p < 0.001; ���� , p < 0.0001).

TNBC cells induce tumor-associated macrophages with characteristics of a mixed M1 and M2 phenotype
TAMs play critical roles in tumor progression. In TNBC, the infiltration of macrophages in the tumor tissue promotes disease progression and leads to a poor prognosis [19]. To examine the crosstalk between TAMs and TNBC cells, the human monocytic leukemic cell line THP-1 was used to generate an in vitro model of TAMs. THP-1 cells were differentiated into macrophages by PMA stimulation for 24 h, followed by treatment with CM obtained from two TNBC cell lines, namely, MDA-MB-231 and MDA-MB-468, to generate TAMs, as described in Fig 1A. As previous observations suggested that most TAMs closely resemble M2 macrophage, PMA-stimulated THP-1 cells were polarized into M2 macrophages with IL-4 and IL-13, as previously described [20]. This population served as a positive control for the comparison of THP-1-differentiated TAMs induced by TNBC-CM. Through flow cytometry, we analyzed the expression of CD163, a surface marker expressed on M2 macrophages. Consistent with previous reports [8], TAMs generated by incubation in TNBC cell CM expressed significantly higher CD163 levels than THP-1-derived macrophages (control). The percentage of CD163+ cells in TNBC-induced TAMs was similar to those of M2-polarized macrophages using IL-4 and IL-13 (Fig 1B and 1C). We then quantified the mRNA level of classical M1 markers (CXCL10, IL-1β, and TNF-α) and alternative M2 markers (TGF-β1, TGF-βR2, CCL22, CCL18, and IL10) relative to THP-1-derived macrophages (control). Cytokine-polarized M2 macrophages demonstrated a significant upregulation of CCL18 and modest expression of M1-associated genes, confirming the characteristics of the M2 phenotype (Fig 2A and 2B). Interestingly, MDA-MB-231-polarized TAMs exhibited a distinct gene expression profile of M2 phenotype with upregulation of TGF-β1 but no increase in CCL18. Besides, an abundance of M1 associated gene expression including CXCL10, IL-1β, and TNF-α were observed (Fig 2A and 2B). The elongation of macrophages correlates with a reduction of proinflammatory cytokines and polarization of macrophages toward the M2 phenotype [21]. In this study, we observed that M2-polarized THP-1 exhibited pseudopodia formation, whereas unstimulated THP-1 and THP-1-derived macrophages displayed a large, round, single-cell morphology ( Fig 2C). However, MDA-MB 231-induced TAMs displayed a mixed morphology of rounded and elongated cells. Moreover, analysis of cytokines secretion revealed that M2-polarized macrophages secreted high levels of IL-4 and low levels of IL-6 and TNF-α, which are M1 mediators that stimulate a proinflammatory immune response [22]. Unlike M2-polarized macrophages, TNBC-induced TAMs secreted a substantial level of IL-10, IL-6, and TNF-α, while IL-4 was barely detected (Fig 2D). Taken together, the results of gene expression, cell morphology, and cytokines secretion have demonstrated that TNBC may induce overlapping and mixed populations of THP-1 derived TAMs. In addition, the mixed M1/M2 phenotype was also observed in primary monocytes that were treated with CM from two different TNBC cell lines; MDA-MB-231 and MDA-MB-468, as indicated by an upregulation of both M2 (CD206) and M1 (CD282, CD284) surface markers (S1 Fig).

TNBC-induced TAMs increase breast cancer cell proliferation and migration
In the TNBC microenvironment, tumor stromal cells including macrophages secrete several factors that can promote tumor growth and metastasis [23]. As TNBC-induced TAMs exhibited M1/M2 phenotype heterogeneity and produced the tumor-promoting cytokine IL-6, we sought to evaluate the effects of TNBC-induced TAMs on TNBC growth. THP-1-derived macrophages, M2-polarized macrophages, or MDA-MB-231-induced TAMs were co-cultured with MDA-MB-231 in a Transwell system that allowed the exchange of soluble factors ( Fig  3A). After 72 h of co-culture, MDA-MB-231 cells were subjected to WST-1 assay to ascertain their proliferation rate. As displayed in Fig 3B, TAMs with a mixed M1/M2 population significantly enhanced the proliferation of TNBC cells, eliciting a fold change similar to that of M2-polarized macrophages.

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Then, we determined whether TAMs could promote TNBC cell migration, which is a critical step in tumor metastasis. We used a wound-healing assay to determine the total wound area at each time point, as previously described [15]. The migratory activity of MDA-MB-231 was measured at 6, 12, and 24 h after the infliction of a scratch wound to exclude the cell proliferation effect (Fig 4A). TAMs generated from MDA-MB-231-CM and cytokine-polarized M2 macrophages significantly increased the mobility of TNBC cells (Fig 4A and 4B). Thus, our results suggest that TAMs with a mixed M1/M2 phenotype could promote cell proliferation and migration in TNBC cells similar to that of IL-4/IL-13-polarized M2 macrophages.

TNBC but not HR + BCA secrete high amounts of IL6
A study revealed that BCA with different molecular characteristics distinctly influenced macrophage phenotype and function [10]. Thus, we sought to investigate the phenotype of TAMs induced by hormone receptor-positive (HR + ) BCA. We observed that TAMs polarized by CM collected from HR + BCA (MCF-7 cells) demonstrated an upregulation of M2-associated genes, including TGF-b1 and CCL22. However, M1-associated proinflammatory mediator

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expressed gene profile (Fig 5B). These results support previous evidence that TNBC regulated distinct macrophage phenotype and biological response.
Then, we determined the cytokine secretion profile in TNBC and HR + BCA cell lines. Cultured supernatants from MDA-MB-231, MDA-MB-468, and MCF-7 cells were collected and the levels of IL-4, IL-10, IL-6, and TNF-α were quantified. MDA-MB-231 cells secreted large amounts of IL-6 compared with MDA-MB-468 and MCF-7 cells. The level of secreted IL-6 was also significantly higher in MDA-MB-468 cells than in HR + MCF-7 cells. We found that the levels of IL-4, IL-10, and TNF-α were not significantly different among the three cell lines (Fig 5C). To address the possibility that a high IL-6 level may contribute to the M1/M2 mixed phenotype of TAMs educated by MDA-MB-231, we polarized PMA-stimulated THP-1 with MCF-7-CM in the presence of rhIL-6 at 1000 pg/mL and quantified the mRNA level of related genes. We hypothesized that the addition of exogenous rhIL-6 may result in the upregulation of proinflammatory cytokines in MCF-7 induced TAMs. However, only a significant reduction of CCL22 gene expression with the addition of IL-6 in the MCF-CM polarized condition was observed, but the mRNA expressions of M1-associated genes including CXCL10, IL1B, and TNF were not different between MCF-7-TAMs and MCF-7-TAMs supplemented with rhIL-6.

M1-associated genes are upregulated in human TNBC tissues
Collectively, our data demonstrate the upregulation of M1-associated genes in TNBC induced TAMs but not in the HR + BCA cells. To consider whether this phenomenon correlate to breast cancer environment in humans, differential gene expression analyses of M1-and M2-associated genes were analyzed in TNBC versus non-TNBC tissues from patients using two independent public databases, METRABRIC and TCGA (Fig 6A). Three M1-associated genes, including CXCL10, IL1B, and TNF were significantly upregulated in TNBC tissues in both datasets. Additional cytokine genes including IL-4, IL-10, and IL-6 were also analyzed. In consistency with the in vitro result, IL6 showed significantly high level in TNBC comparing to non-TNBC tissues. Furthermore, we performed Kaplan-Meier survival analyses of patients bearing high versus low levels of M1-associated genes (Fig 6B). Interestingly, high CXCL10 level was significantly associated with shorter disease-free period, whereas high IL1B level was associated with delayed disease progression. Univariate and multivariate analyses were performed with tumor stage and cancer type as covariates ( Table 1). The results showed that expression levels of CXCL10 and IL1B were significantly prognostic of disease-free survival independently of other clinical factors.

Discussion
In the tumor microenvironment, TAMs, originating from blood monocytes, are recruited to tumor tissues by factors secreted by tumor cells and the tumor stroma and constitute the majority of tumor-infiltrating immune cells. Given the high plasticity and diversity of myeloid-lineage cells, the tumor milieu, depending on the location, tumor type, and local stromal cells, shapes the polarization and biological function of TAMs [24][25][26]. By contrast, TAM infiltration is a poor prognostic factor and associated with aggressive tumor characteristics [27][28][29]. This crosstalk between cancer cells and TAMs is widely validated in ex vivo experiments where human monocytic leukemic cells (THP-1) were co-cultured with cancer cells or cancer CM.
THP-1 monocytes can be differentiated in vitro using PMA into macrophage-like cells that resemble functional mature macrophages [30][31][32]. Genin et al. reported that PMA-differentiated THP-1 can be polarized into M1 or M2 macrophages with expression profiles similar to those of polarized primary monocytes. Moreover, these THP-1 M2-like macrophages prevented an apoptotic response to etoposide in two cancer cell lines, namely, HepG2 and A549 [20]. Multiple evidence also indicates that CM from cancer cells regulate the differentiation of THP-1 monocytes into tumor-promoting M2-like macrophages [33,34].
In breast cancer, THP-1 treated with CM from MCF-7 or MDA-MB 231 cells results in the differentiation of THP-1 cells toward macrophages and increases the mRNA expression of M2-type macrophage markers, including CD163 and IL-10 [35]. In this study, we consistently reported that CM from MDA-MB-231 cells induced PMA-stimulated THP-1 to express  CD163 on the cell surface and upregulate the immunosuppressive cytokine TGFβ1 and its cognate receptor TGFβR2. In contrast to THP-1-derived macrophages polarized by IL-4/IL-13, TNBC-induced TAMs demonstrated high expressions of M1-associated genes, including that of CXCL10, IL-1b and TNF-a. We also observed less pseudopodia formation in TNBC-induced TAMs than in IL-4/IL-13-polarized M2 macrophages. Our results indicate that soluble factors secreted by TNBC cells induce the polarization of THP-1-derived macrophages into a distinct TAM population with defining characteristics of M1 and M2 types. This phenomenon appears to be preserved in TNBCs because we did not observe the upregulation of M1-associated genes in HR + MCF-7 induced TAMs. This is in agreement with the finding of a previous study that THP-1 cells exposed to basal-like breast cancer cells in a co-culture system exhibit the upregulated gene expression of both M1 and M2 macrophage markers [36].  [10]. The mixed polarization of M1/M2 type in THP-1 treated with tumor-soluble factors was also observed in colorectal cancer, in which CM from several colon cancer cell lines enhanced the phagocytic activity of THP-1 cells [25] and induced the production of cytokines and chemokines typical of both M1 and M2 macrophages, including IL-1β, MCP-1, IL-6, and IL-10 [37]. At the protein level, we reported that TNBC-induced TAMs but not M2-polarized macrophage secrete large amounts of IL-6 and TNF-α. The abundance of IL-6 and TNF-α was comparable between MDA-MB-231 and MDA-MB-468 cells. TNBC-induced TAMs also secrete high levels of IL-10 but not IL-4. A study revealed that TAMs infiltrating TNBCs secrete IL-10, which contributes to tumor progression [38]. In our study, M2-polarized macrophages secreted significant amounts of IL-4, which are proposed to possess anti-inflammatory and tumor-promoting properties [39]. However, only a small amount of IL-10 in the culture supernatant of M2-polarized macrophages was detected. We examined the CM of MDA-MB-231 and detected a very small amount of IL-4. This absence of IL-4 in MDA-MB-231-CM could be attributed to the different phenotypes of TNBC-activated TAMs from M2-macrophages polarized by IL-4/IL-13. Chan et al. analyzed cytokine expression using MILLIPLEX assay in human breast cancer cell lines and reported that MDA-MB-231 cells secreted moderate and low amounts of IL-4 and IL-13, respectively [40].
In line with previous findings [40], TNBC cell lines significantly secreted higher amounts of IL-6 than did the HR + BCA cell line. The magnitude of IL-6 production by TNBC cells is correlated with aggressive behavior, where MDA-MB-468, a less aggressive cell line, secretes a much lower level of IL-6 than MDA-MB-231. Several tumor cells, including breast cancer cells, can secrete IL-6, the upregulation of which is generally associated with poor prognosis and a low survival outcome [41,42]. Previous evidence also revealed IL-6 promotes TNBC cell survival [43], increases breast cancer stemness [44], and contributes to chemoresistance of MDA-MB 231 cells [45]. Furthermore, primary macrophages secrete IL-6, which in turn increase IL-6 secretion from cancer cells [46], where IL-6 is shown to perpetuate this vicious cycle by generating more aggressive M2 macrophage polarization by activating Stat3 phosphorylation [44,47]. In the present study, we investigated the influence of IL-6 on the distinct characteristics of TNBC-induced TAMs by the generation of TAMs with MCF-7-CM-containing rhIL-6 at a similar concentration found in the MDA-MB-231-CM. However, the addition of IL-6 did not upregulate M1-associated genes in MCF-7-induced TAMs, suggesting that multiple mediators contribute to the complexity of macrophage polarization induced by breast cancer. Interestingly, the incorporation of IL-6 downregulates CCL22 in MCF-7-induced TAMs. CCL22 is expressed in alternative (M2) macrophage [48,49] and in many types of cancer [50][51][52]. The functional role of CCL22 in cancer immunity involves recruiting Tregs to the tumor stroma by binding to its cognate receptor CCR4 [53,54]. A previous study suggested that CCL22 is downregulated by the TH1 cytokine IFNγ [55]. The mechanistic interaction between IL-6 and CCL22 in TNBC should be explored for further investigation. Future experiments such as the incorporation of IL-6 neutralizing antibody or the deletion of IL-6 with siRNA could be necessary to determine the role of IL-6 in the polarization of TNBC-educated TAMs.
Finally, the results from in vitro experiments were validated by analyses of two independent patient databases that showed a significant greater M1-associated genes expression in TNBC than non-TNBC. The expression level of IL6 was also significantly higher in TNBC compared with non-TNBC tissues, whereas IL4 level was not different between cancer types. This result supported our finding that TNBC cells secreted large amounts of IL-6 comparing to non-TNBC cells. Interestingly, the expression of CXCL10 have strong prognostic value for diseasefree survival in the breast cancer human cohort, suggesting a critical contribution of this molecule in breast cancer outcomes.
In summary, our results demonstrate that TNBC cells but not HR+ cells induce a distinct population of M2-like TAMs that express M1-associated genes and secrete a considerable amount of IL-10 and IL-6, which in turn promotes breast cancer cell proliferation and metastasis. Although these TNBC-induced TAMs exhibit a differential gene expression profile, their tumor-promoting effects were similar to those of M2 macrophages polarized by IL-4 and IL-13. One of the limitations in this study is the lack of M1 and M2 associated enzymes and proteins analyses. Nevertheless, the M1/M2 mixed phenotype on TNBC-induced TAMs were demonstrated by gene expression, cellular morphology, and cytokines profile of THP-1 derived macrophage as well as surface expression of M1 and M2 markers on primary monocytes. Moreover, analysis of patient database follows the in vitro finding where M1 associated genes are upregulated in TNBCs particularly the key marker CXCL-10 which is negatively correlated with disease-free survival. Together, our results suggest the complexity and heterogeneity of TAMs and their negative roles in TNBC.